Method for making stable, extracellular tyrosinase and synthesis of polyphenolic polymers therefrom

ABSTRACT

A process for the in vitro production of chemically modified polyphenolic polymer (PPP). First, stable, highly active extracellular tyrosinase is produced from genetically transformed microorganism such as Streptomyces antibioticus. The tyrosinase is then incubated with a reaction substrate such as l-tyrosine, hydrolyzed protein, or an oligopeptide in combination with l-tyrosine. The ratio of the oligopeptide/tyrosine combination as well as variation in the concentration of tyrosinase can be used to modify the color, the molecular size, and the spectral absorbance properties of the PPP produced. Alternatively, or additionally, oxidants such as hydrogen peroxide or hypochlorite can be used to modify the color of the PPP, regardless of the method used to produce the PPP, and the PPP can subsequently be fractionated using molecular weight cut-off ultrafiltration. Organic solvents can also be used in the method of making PPP to produce PPPs having variable but reproducible physical properties.

This is a divisional of application Ser. No. 07/982,095, filed Nov. 25,1992, now U.S. Pat. No. 5,340,734.

BACKGROUND OF THE INVENTION

Melanin is an omnibus term that describes a large family of natural andsynthetic phenolic-quinonoid pigments of diverse origin and chemicalnature. Natural melanins are generally differentiated by their origin,for example, bovine eye melanin, melanoma melanin, and sepia melanin.They usually occur in the form of granular particles and are secretoryproducts of pigment-producing cells, the melanocytes. Synthetic melaninsare named after the compound from which they were prepared via chemicalor enzymatic oxidation (e.g., d,l-dopa., or 5,6-dihydroxyindole catecholmelanin). In addition, melanins are classified according to theirchemical structures into the insoluble black eumelanins (poly-5,6-indolequinones) and the alkali soluble red phaeomelanins(polydihydrobenzothiazines). The study of melanins has led to thediscovery of a number of biosynthetic pathways. For example, melaninscan be produced by the oxidation of its precursors such as l-dopa ortyrosine by a melanin-synthesizing enzyme, tyrosinase. Alternatively,melanin can be prepared chemically by auto-oxidation of l-dopa or othersubstrates to melanin in the presence of atmospheric oxygen. Wilczok etal., Arch Biochem. Biophys. 23: 257 (1984). Additionally, melanin can beprepared by a variety of electrochemical and photochemical methods fromwhich individual steps of the melanization processes are identified andcharacterized. See, Crippa, et al. (1989), supra.

When melanin shows a characteristic UV absorption spectrum at aparticular range of wavelengths, it is assumed that the melanin mayexert a protective effect by absorbing or diminishing the energygenerated by the correspondent wavelengths. In general, sunlightcontains three types of ultraviolet wave lengths which may cause skindamage. UV A has the longest wave length and the lowest energy. Itswavelengths fall within the range of 320-400 nm, and such wavelengthscan indirectly damage DNA by activating intracellular flavins orporphyrins in the formation of active oxygen species, e.g., hydrogenperoxide, hydroxyl radicals, oxygen radicals, etc. UV B falls within therange of 290-320 nm. It damages DNA both directly and indirectly. Thistype is probably the major causative factor in skin cancer. UV C haswavelengths between 200-290 nm. Its major effect is directly related todamage of cellular DNA.

Characteristics of light relevant here fall into two major categories:(1) luminous flux which is the effectiveness of light evokingbrightness; (2) chromaticity which is referred to both the dominantwavelength that contributes to the actual color (i.e., hue) and thepurity that establishes the saturation of color. The spectrum colors andtheir wavelengths are further categorized as the follows: violet (400nm), blue (450 nm), green (500 nm), yellow (550 nm), orange (600 nm) andred (650 nm).

The enzymatic synthesis of melanin has been an area of extensiveresearch due to its close resemblance to natural melanogenesis. Theenzymes, tyrosinases, are widely distributed in nature and highlypurified preparations have been obtained from mushrooms, (Lerch, K.,Met. Ions. Biol. Syst. 13: 144 (1981)), Neurospora crassa, Podosporaanserina, potato tubers, broad beans, insect hemolymph and mammalianmelanoma tumors. Lerch, K. and L. Ettlinger, Eur. J. Biochem, 31:427-437 (1972). It is generally agreed that these enzymes catalyze twotypes of reactions: the orthohydroxylation of monophenols to catechols,which is referred to as cresolase activity, and the dehydrogenation ofcatechols to o-quinones, designated as catecholase activity. Molecularoxygen is used for the hydroxylation reaction. For this reason,tyrosinase acting on a monophenol is referred to as a "mixed functionoxidase". Hayaishi, O. in "Biological Oxidation" (Singer, T. P., ed.)p.581, Interscience Publishers, New York (1968).

Numerous investigations have revealed that the production of tyrosinaseby a microorganism in a growth medium is regulated by such factors asthe genetics of the microorganism, the composition of the medium, thegrowth temperature, the presence of biosynthetic inhibitors, the densityof tyrosinase-producing cells and the presence of enzyme inducers. Katz,E. and A. Betancourt, Can J. Microbiol, 34: 1297-1303 (1988). Somemicroorganisms are capable of producing extracellular tyrosinases whichare synthesized intracellularly prior to their transport and secretioninto the growth medium. Baumann, R., et al., Actinomycetes "The BoundaryMicroorganisms", pp.55-63, (Arai, T. ed), Tokyo Joppan Co. (1976).

The discovery of extracellular tyrosinase makes industrial pigmentproduction by fermentation feasible. However, the high instability ofthe extracellular tyrosinase in the broth limits the recovery of activetyrosinase in these processes and hence limits production of melanin.Bauman, R. et al., (1976), supra. Moreover, the quantities and recoveryof the secreted extracellular tyrosinases have been generallyinsufficient for a viable commercial process for the large-scale invitro production of melanin. Loss of enzyme activity as a result ofdeactivation through the affects of oxygen, temperature and productintermediates has a severe affect on the yield of tyrosinase and,therefore, is responsible for the high instability and low yield ofextracellular tyrosinase. Studies in Streptomyces antibioticus haveshown that the kinetics of the loss of tyrosinase activity could beapproximated by a first-order model, with a constant specificdeactivation rate on the order of 0.1 h⁻¹. Proteolytic activity detectedin stationary phase cultures suggested that proteolytic degradation oftyrosinase could be partly responsible for the loss of tyrosinaseactivity during this period of batch cultures, but tyrosinasedeactivation was also observed during the growth phase when noproteolytic activity was detected. The specific deactivation rate wasfound to exhibit an Arrhenius dependence on temperature with anactivation energy of approximately 20 kcal/mol. Growth of a cultureusing a two-temperature strategy along with an enriched growth mediumresulted in a 2.5-fold increase in the amount of tyrosinase obtainedover control cultures. Gardner, A. R. and T. W. Cadman, Biotechnologyand Bioengineering 36: 243-251 (1990). Improved processes for makingtyrosinase and melanin are disclosed in PCT publication W092/00373 datedJan. 9, 1992, which claims priority from U.S. Ser. Nos. 545,075, Jun.29, 1990 and 607,119, Nov. 2, 1990, the disclosures of which are eachincorporated by reference herein.

In addition to growth manipulation and temperature optimization, severalmicroorganisms may be genetically engineered to further enhance theirabilities to produce tyrosinases. These microorganisms include, but arenot limited to species of Streptomyces, Escherichia, Bacillus,Streptococcus, Salmonella, Staphylococcus, and Vibrio. Many species ofStreptomyces are capable of forming dark melanin pigments due toexpression of tyrosinase from the mel gene locus. For example, the mellocus of S. antibioticus has been cloned and sequenced, Katz, E., etal., J. Gen. Microbiol. 123: 2703 (1983); Bernan, V. et al., Gene 37:101 (1985) and shown to contain two open reading frames (ORFs) thatencode a putative ORF438 protein (MW=14,754) and tyrosinase (MW=30,612).ORF438 and tyrosinase are thought to be transcribed from the samepromoter in S. antibioticus, and both genes are required for melaninproduction. Bernan, V., et al., (1985), supra. Based on geneticevidence, the ORF438 protein has been shown to function as atrans-activator of tyrosinase. Lee, Y.-H. W. et al., Gene 65:71 (1988).It has been suggested that the ORF438 protein is involved in tyrosinasesecretion, or it may function as a metallothionein-like protein thatdelivers copper to apotyrosinase. Bernan, V., et al., ( 1985), supra;Lee Y.-H. W., et al., (1988), supra.

The oxidation of a variety of N-terminal, C-terminal, and internaltyrosine peptides of melanin in the presence of mushroom tyrosinase hasbeen studied spectrophotometrically. The spectroscopic patterns of theseoxidations fall into three types: a dopachrome sequence, a dopa-quinonesequence, and a protein sequence. The dopachrome pattern ischaracterized by the formation of an intermediate oxidation product withabsorption maxima at 305 nm and 480 nm. This absorption spectrum isexhibited by compounds of the aminochrome class, especially bydopachrome itself. The final oxidation product shows a maximum at 325nm. This is very similar to the maximum at 319 nm exhibited byderivatives of 2-carboxy-5,6-dihydroxyindole, which is an intermediatein the enzymatic oxidation of 3,4-dihydroxyphenylalanine underconditions in which decarboxylation does not take place. Compounds whichshow the dopachrome pattern upon oxidation have blocked carboxyl groupsand cannot undergo decarboxylation. It is accordingly probable that thedevelopment of the 325 nm absorption band is due to the accumulation ofa derivative of 2-carboxy-5,6-dihydroxyindole. The dopaquinone patternis characterized by the formation of an intermediate oxidation productwith an absorption maximum at 390 nm. The protein pattern of oxidation,which is observed with a polypeptide which contains tyrosine may vary,depending upon the particular substrate. The distinctive absorptionpatterns, therefore, may be used to evaluate the positions of tyrosinein the peptide chains. Yasunobu, K. T., et al., J. Biol Chem.234:3291-3205 (1959).

Similar spectrophotometrical studies have been conducted on melanin forthe enzymatic oxidation of a single amino acid in the presence oftyrosinase. The process was observed to proceed in three chromophoricphases, the first corresponding to the formation of the red pigment, thesecond to an intermediate purple pigment, and the third to the formationof melanin. By comparison of the observed spectra of these products withthose of known substances, it is possible to identify the intermediatesduring the process of melanin formation. Mason, H. S., J. Biol. Chem.,168: 433 (1947); Raper, H. S., Biochem, J., 21: 89-96 (1927).

Recently it has been shown that a number of enzymes can express theiractivity in reaction media where most of the water has been replaced byan organic solvent. Kibanov, A. M. Chemtech June, 354-359 (1986). Forexample, laccase purified from Trametes versicolor can oxidize2,6-dimethoxyphenol and syringaldazine in hydrophobic solventspresaturated with water. Milstein et al., Appl. Microbiol. Biotechnol31: 70-74 (1989). In addition, the ortho-hydroxylation of aromaticcompounds by the enzyme polyphenol oxidase such as tyrosinase suspendedin organic solvents has also been found to be efficient. Doddema, H.J.Biotechnology and Bioengineering 32: 716-718 (1988). However, some ofthe reported prior art methods require the enzymes to be immobilized onan inert surface or carrier prior to enzymatic reaction conducted inorganic solvents.

In addition to tyrosinase, a number of other enzymes possessing aperoxidizing activity such as horseradish peroxidase, chloroperoxidase,milk peroxidase, cytochrome C peroxidase and microperoxidase have alsobeen used to produce melanin in vitro. These methods generally requirethe inclusion of hydrogen peroxide as a substrate in the reactionmixture and, in some instances, the immobilization of peroxidase priorto the oxidation reactions. European Patent Publication No. 441, 689 A1.However, the in vitro synthesis of melanin under such harsh conditionshas not been described or suggested when tyrosinases are used as theenzyme sources.

In light of the foregoing, there is a need for an improved method forthe production of stable and highly active extracellular tyrosinase incommercially acceptable quantities. There is a further need to developan improved in vitro process for the synthesis of melanins (hereafterpolyphenolic polymers or "PPPs") generally and chemically modified PPPsspecifically in the absence of a fermentation medium and microorganisms.This need is driven by the advantages that such an in vitro processwould provide, i.e. eliminating the concern about using precursers inthe reaction which would be toxic if used in vivo, and the avoidance ofcomplications arising from the presence of cellular metabolites anddebris.

SUMMARY OF INVENTION

To achieve this end, a first aspect of the present invention relates toa novel method for the in vivo production of commercial quantities of astable, highly active, extracellular tyrosinase. Any transformedmicroorganism which contains an expression vector containing geneticsequences encoding for tyrosinase may be used, so long as themicroorganism is capable of expressing and secreting functionaltyrosinase into the media. The method further comprises the steps ofcausing the microorganism to express extracellular tyrosinase, andrecovering the extracellular tyrosinase from the cellular debris byfiltration or centrifugation.

A second aspect of the invention relates to methods for stabilizing theextracellular tyrosinase so produced by means of low temperaturestorage, with or without a preservative and/or using an inert gas toreplace air surrounding the tyrosinase, or freeze drying. Because thetyrosinase can be stored for extended periods of time, such as for morethan a year, the method provides a particularly useful means forproviding the necessary availability of large quantities of tyrosinaseneeded for a variety of end uses as discussed in detail below.

In a third aspect of the invention, PPPs are produced withoutfermentation, in either aqueous or organic solutions, using tyrosinasemediated polymerization of tyrosine, hydrolyzed proteins, tyrosinederivatives, and tyrosine-containing oligopeptides including tyrosinedipeptides and mixtures thereof. Since the color of PPPs varies withchemical and physical characteristics such as the UV and visibleabsorbance spectra, molecular size, aqueous solubility and substratechemistry, such PPPs may possess different reproducible characteristicssuch as different colors.

In a fourth aspect of the invention the color of the PPP made from thesame substrate/l-tyrosine combination can be modified by varying theratio of each substrate/l-tyrosine combination. Similarly, the color ofPPP that derives from the same substrate/l-tyrosine combination can bevaried to produce different colors substrate/l-tyrosine combination withdifferent concentrations of tyrosinase.

In a fifth aspect of the invention, the color of such PPPs (whetherproduced by the method noted or other known methods) can be modified byuse of a strong oxidizing reagent, such as hydrogen peroxide,hypochlorite, peracetic acid, perchloric acid, or potassium permanganate(Wolfram et al. (1970) J. Soc. Cosmet. Chem. 21: 875-900), andsubsequently recovered by ultrafiltration.

In light of this, the present invention is, in its broadest form, an invivo method of producing a stable extracellular tyrosinase, comprisingthe steps of growing a transformed microorganism which contains anexpression vector containing genetic sequences encoding for tyrosinaseunder suitable growth media and conditions; causing the microorganism toexpress extracellular tyrosinase; and recovering the extracellulartyrosinase from the cellular debris by filtration or centrifugation.

In a more specific form, the method can include the further step ofstabilizing the recovered extracellular tyrosinase by freeze-drying thetyrosinase, or by storing the tyrosinase at a temperature of 22° C. orless, or preferably at a temperature of 4° C. or less, using aprotectant and, if desired, in an inert gas atmosphere.

The method of growing the transformed microorganism can include the stepof preparing an expression vector, where the vector used is the plasmidpBS1082S. The host microorganism for the method can be selected fromStreptomyces, Escherichia, Bacillus, Streptococcus, Salmonella,Staphylococcus, and Vibrio, and is preferably Streptomyces, and mostpreferably Streptomyces antibioticus IMRU 3720.

During the process of making tyrosinase controlling the amount ofdissolved oxygen in the mixture can be used to cause the microorganismto express extracellular tyrosinase in a timely and efficient manner.

The invention further relates to an in vitro method of producingpolyphenolic polymers ("PPPs"), and the PPPs made from that method,comprising the steps of contacting extracellular tyrosinase and areaction substrate selected from the group consisting of l-tyrosine,hydrolyzed protein, X/l-tyrosine, and l-tyrosine/X, where X is a singleamino acid, a dipeptide or an oligopeptide, under suitable reactionconditions, to form PPP and recovering the PPP so produced. In thismethod the tyrosinase and substrate can be contacted in either anaqueous or an organic solvent, where the solvent is preferably methanol,propanol, ethanol or DMSO.

While making PPP it is preferred to use a dipeptide substrate. Inaddition to varying the substrate to affect the color of the PPP, thecolor of the PPP produced can be varied by varying the amount oftyrosinase present in said reaction vessel relative to the combinedamount of the substrate, or by varying the ratio of the substrate tol-tyrosine. Where the dipeptide tyr-ala is used, it is preferred to usea ratio of the dipeptide to l-tyrosine of from 4:1 to 4:10. Where thedipeptide phe-tyr is used, the ratio of the dipeptide to l-tyrosine ispreferably from 5:1 to 1:1.

The invention further contemplates a method for producing PPP comprisingthe steps of combining PPP with a strong oxidizing agent for a timeperiod selected to produce a PPP having a desired color, and removingthe oxidizing agent from the combination. The oxidizing agent ispreferably hydrogen peroxide, sodium hypochlorite, potassiumpermanganate, peracetic acid or perchloric acid. The oxidizing agent ispreferably removed from the reaction mixture by ultrafiltration, andultrafiltration can also be used to fractionate the PPP by usingselected molecular weight cut-off filters.

The invention further includes fractionated PPPs which have beenrecovered using selected molecular weight cut-off filters.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is the genetic map of plasmid pBS1082S, which is used totransform Streptomyces antibioticus according to the invention.

FIG. 2 shows the relationship between tyrosinase yield and the amount ofdissolved oxygen in the fermentation process.

FIG. 3 shows the rate of in vitro PPP production in a 3 liter reactionvessel with rate of synthesis monitored by the increase of absorbance at400 nm.

FIG. 4 shows the U.V. absorption spectra for several dipeptide/tyrosinecombinations in different ratios.

FIG. 5 shows the U.V. spectra of phe-tyr/tyr whose ratios are varied inreference to different concentrations of tyrosinase.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a process for the in vitro productionof chemically modified polyphenolic polymer (PPP). First, stable, highlyactive extracellular tyrosinase is produced from genetically transformedmicroorganism such as Streptomyces antibioticus. The tyrosinase is thenincubated with a reaction substrate such as l-tyrosine, hydrolyzedprotein, or an oligopeptide in combination with l-tyrosine. The ratio ofthe oligopeptide/tyrosine combination as well as variation in theconcentration of tyrosinase can be used to modify the color, themolecular size, and the spectral absorbance properties of the PPPproduced. Alternatively, or additionally, oxidants such as hydrogenperoxide or hypochlorite can be used to modify the color of the PPP,regardless of the method used to produce the PPP, and the PPP cansubsequently be fractionated using molecular weight cut-offultrafiltration. Organic solvents can also be used in the method ofmaking PPP to produce PPPs having variable but reproducible physicalproperties.

We have found that the physical characteristics of PPP, such as color,may vary with chemical composition. The addition of selected amino acidsor peptides to the reaction mixture during synthesis may result in theproduction of various colors due to their incorporation into the PPPstructure. For example, the incorporation of the single amino acidtyrosine will result in a black PPP. The resultant colors can be furthermodified by chemical oxidation with, but not limited to, hydrogenperoxide or hypochlorite.

PPPs of the present invention have a variety of uses including, but notlimited to, protective effect against UV radiation (as a major componentin sunscreen products), as antioxidants, as antivirals, as a therapeuticagent to treat neurological disorders, as a protectant for plastics, asa chelator for binding materials such as metals and other compounds, asa coloring agent for foods and hair dyes, and other uses such as in theproduction of semiconductors.

The present invention will be better understood by reference to thefollowing definitions of the terminology used throughout thisspecification.

PPPs are polyphenolic polymers having molecular weights ranging from 500to over a million. They are comprised of a basic phenolic backbonestructure which may include phenolic derivatives such as indole. Theymay also include dipeptides in their structure. The PPPs of the presentinvention are polymers formed in a reaction catalyzed by tyrosinase orsimilar enzymes.

Tyrosinase is useful in the production of PPPs such as melanin and forcatalysis of biological adhesives, and other uses. Tyrosinase is anenzyme that catalyzes the orthohydroxylation of monophenols tocatechols, which is referred to as cresolase activity, and thedehydrogenation of catechols to o-quinones, designated as catecholaseactivity. Molecular oxygen is used for the hydroxylation reaction. Thus,tyrosinase may also be useful in bioremediation due to its ability tofunction as an oxidizer and its ability to promote or catalyze suchorthohydroxylation and dehydrogenation.

The tyrosinase of the present invention is typically recovered from acell-free broth which can be further processed by ultra-filtration whichresults in the concentration of the enzyme and the removal of salts andlow molecular weight media and metabolic products. The tyrosinase may beproduced by the method of U.S. Ser. Nos. 545,075, filed Jun. 29, 1990,607,119, filed Nov. 2, 1990, and 857,602, filed Mar. 30, 1992, or by themethod of Gardner, et al, supra. The removal of the cellular debris andthe ultra-filtration allows for the production of a stable tyrosinasethat may be used subsequently in the production of PPPs. Typically theenzyme is stored at -20° C. Alternatively, the enzyme can be freezedried. Various protectants may be used to stabilize the enzyme such asby the addition of polyhydroxy alcohols such as and most preferably 50%glycerol. The levels of these protectants may range from 5-75% byweight, preferably 35-65%. Storage of the enzyme in an inert gas allowsfor longer storage and stability of the enzymes.

Numbers can be used to express the color of synthesized PPPs. One methodin common usage is the so-called CIE 1976, L*a*b* (pronounced L-star,a-star, b-star) color system wherein the numbers that are used tocharacterize the colors are defined as follows: L* is called the "value"which is a measure of the lightness or darkness of an object. a* and b*represent hue on two axes, with a* the red-green axis and b* theyellow-blue axis. A full description of this system is described inBillmeyer and Saltzman Principles of Color Technology, (2nd ed. 1981),which is hereby incorporated by reference.

Tyrosinase Production

A prerequisite for a large-scale in vitro production of PPPs is theavailability of a commercial quantity of a stable, highly activetyrosinase which is subsequently used for the in vitro PPP synthesis.Accordingly the present invention comprises a method for the productionof commercial quantities of a stable, highly active tyrosinase from amicroorganism, preferably, Streptomyces antibioticus. Geneticmanipulation involves the transformation of a microorganism with aplasmid or a vector which contains a gene encoding tyrosinase. Thetransformed microorganism is not only capable of expressing tyrosinase,but also capable of secreting it to the medium. The techniques for theconstruction of plasmids, vectors and the transformation of themicroorganism suitable for the enhancement of tyrosinase production havebeen described in U.S. patent application Ser. Nos. 545,075, filed Jun.29, 1990, 607,119, filed Nov. 2, 1990, and 857,602, filed Mar. 30, 1992,which applications are hereby incorporated by reference. (Thecorresponding PCT application is WO92/00373, published Jan. 9, 1992.)The spores produced by the transformed Streptomyces antibioticus areprepared from ISP4 thiostrepton plates and subsequently inoculated at aconcentration of 2.0×10⁵ CFU/liter media, as shown in example 1(a).

One method of increasing the yield of active tyrosinase is to enhancethe stability of extracellular tyrosinase after it has been synthesizedand secreted by the microorganisms into the media. We have found thatour novel low casein media for enzyme production results in a reducedbiomass providing a significantly improved, more efficient, enzymerecovery. Applicants have further found that precise control ofdissolved oxygen during fermentation results in a increased tyrosinasestability during production (see FIG. 2). The low casein media used forthe in vivo production of tyrosinase from the transformed Streptomycescomprises the combinations of appropriate volumes of components providedin example 1(a). Each of the media components is added timely andsequentially to the media to ensure the maximum production oftyrosinase. As can be seen in FIG. 2, a decrease of 5.8% of dissolvedoxygen per hour between 24 and 39 hours of the fermentation by adjustingthe air flow rate and agitation resulted in the production of 65.9units/ml tyrosinase. The cells are removed from the broth by filtrationand centrifugation and the resultant cell-free broth containingtyrosinase may be used as is or stored at -20° C. until needed forsubsequent in vitro PPP synthesis or other uses. The extracellularstable tyrosinase activity is monitored through the fermentationspectrophotometrically by the dopachrome method (Yasunobu, K. T., etal., J. Biol Chem. 234: 3291-3205 (1959)). As a consequence of thesemanipulations, the invention provides a method to produce extracellular,stable tyrosinase at a quantity of over 60 units/ml (see FIG. 2).

Once the tyrosinase is formed, it is stabilized and stored, if desired,for later use. When it is collected as a cell free broth, it can bestored at low temperature, e.g. 4° C. or less and preferably at -20° C.or less. To extend the storage time the broth may be exposed to an inertgas (by sparging to remove air), such as argon. A protectant, such asglycerol can be added to further increase storage time. Alternatively,the tyrosinase can be freeze dried.

EXAMPLE 1(a) Production of Stable, Highly Active Streptomyces Tyrosinase

Streptomyces antibioticus IMRU 3720, obtained from the NRRL, strain(B-16570), was transformed with a constructed plasmid pBS1082S ofFIG. 1. Spore stocks were prepared from ISP4 thiostrepton plates (50mg/ml) as described in Hopwood et al., Genetic Manipulation ofStreptomyces, A Laborabory Manual, The John Innes Foundation, F. Croweand Sons Ltd., Norwich, England (1985), the contents of which areincorporated by reference herein.

The spore stock production media used is provided below:

    ______________________________________                                        ISP4 + Thiostrepton Plates                                                    ______________________________________                                        g/liter                                                                       10.0             Soluble potato starch                                        1.0              K.sub.2 HPO.sub.4                                            1.0              M.sub.g SO.sub.4.H.sub.2 O                                   1.0              NaCl                                                         2.0              (NH.sub.4).sub.2 SO.sub.4                                    2.0              CaCO.sub.3                                                   20.0             Agar                                                         0.5              l-tyrosine                                                   1.0     ml/liter Mineral salts                                                1.0     ml/liter 0.5% CuSO.sub.4.5H.sub.2 O (as below)                        1.0     liter    Deionized H.sub.2 O                                          ______________________________________                                    

The media was autoclaved for 20 minutes and then cooled to 50° C. One mlof 50 mg/ml thiostrepton was added into the media. One liter ofISP4+thiostrepton was sufficient to make forty 8.5 cm diameter roundpetri plates or five 506 cm² square plates.

The copper sulfate solution used in the above formulation is prepared asfollows: One-half gram of CuSO₄.5H₂ O is used in 100 ml deionized H₂ O,and then autoclaved for 20 minutes for spore stocks.

    ______________________________________                                        Mineral Salts Solution                                                        ______________________________________                                        mg/liter                                                                      40        mg     ZnCl.sub.2                                                   200       mg     FeCl.sub.3.6H.sub.2 O                                        10        mg     CuCl.sub.2.2H.sub.2 O                                        10        mg     MnCl.sub.2.4H.sub.2 O                                        10        mg     Na.sub.2 B.sub.4 O.sub.7.10H.sub.2 O                         10        mg     (NH.sub.4).sub.6 Mo.sub.7 O.sub.24.4H.sub.2 O                1         liter  Deionized H.sub.2 O                                          ______________________________________                                    

The solution was autoclaved for 20 minutes.

Thiostrepton Stocks

Thiostrepton stocks were prepared by dissolving 50 mg thiostrepton into1.0 ml DMSO. Antibiotic stocks were stored at -20° C. until needed.

Twenty liters of low casein production media was inoculated with 2.0×10⁵CFU/liter media in a 30 liter Braun fermentor. The fermentationproceeded at 30° C., 320 rpm and 4.0 liters per minute air flow. Anadditional 160 g Marcor casein peptone type M in 900 ml water and 6 mlpeanut oil was added to the fermentation at 23 hours post inoculation.At 24.5 hours post inoculation, the temperature was reduced from 30° C.to 25° C. At 26 hours post inoculation, 1.0 ml of 5.0% CuSO₄.5H₂ O wasadded to the fermentor. A constant decrease of 5.8% dO₂ /hr wasmaintained between 24 and 39 hours of fermentation by adjusting the airflow rate and agitation (see FIG. 2). Extracellular tyrosinase activitywas monitored throughout the fermentation spectrophotometrically by thedopachrome method, where unit activity is defined by Lerch and Ettlinger(1972): one unit of tyrosinase will convert one mole of l-dopa todopachrome in one minute at 30° C. and pH 6.0. When the extracellulartyrosinase activity stopped increasing at 39.5 hours, (65.9 units/ml),the culture was harvested.

The fermentor was cooled to 17.4° C. Cells were removed from thefermentation broth by filtration through cheese cloth followed bycentrifugation with an Alpha Laval centrifuge. The broth was maintainedat 4° C. during processing. The cell-free broth can be used for in vitroPPP production and for other uses as is or can be stored at -20° C. forlater use.

Copper Sulfate Solution

In the low casein production media, 5 grams of CuSO₄.5H₂ O per 100 ml ofH₂ O is prepared and then autoclaved for 20 minutes.

    ______________________________________                                        Low Casein Production Media                                                   ______________________________________                                        grams/liter                                                                   4.0     g      Marcor Casein Peptone Type M                                   1.0     g      KH.sub.2 PO.sub.4                                              0.5     g      MgSO.sub.4.7H.sub.2 O                                          1       liter  Deionized H.sub.2 O                                            ______________________________________                                    

Marcor Casein Peptone Type M Addition for 20 Liters

One-hundred sixty grams of Marcor Casein Type M were dissolved in 900 mldeionized H₂ O, with 6 ml peanut oil and then autoclaved for 35 min.

EXAMPLE 1(b) Tyrosinase Recovery

Tyrosinase from two 20 liter Braun fermentations was concentrated 25fold to a 1.2 liter total volume by filtration through a Setec spiralU.F. membrane with a 10,000 molecular weight cutoff. The concentratedtyrosinase was diafiltered 2 times with 2 liters of distilled water andstored at -20° C. until needed in this case for in vitro PPP synthesis.

EXAMPLE 1(c) Tyrosinase Stabilization

Tyrosinase prepared as in example 1(a) and 1(b) can be stabilized by avariety of techniques. A number of methods were examined and thetyrosinase activity half-life was determined for each storage method(see Table 1). Cell-free broth was stored at different temperatures andin the presence or absence of 50% glycerol. Air was removed from somepreparations by sparging the tyrosinase storage container with argon. Inaddition, ultra-filtered tyrosinase was lyophilized and stored atdifferent temperatures. The tyrosinase half-life activity was determinedby periodically sampling each preparation and measuring the remainingtyrosinase activity by the dopachrome method.

                  TABLE 1                                                         ______________________________________                                                                     Tyrosinase                                       Enzyme         Storage       Activity                                         Preparation    Temperature   Half-Life                                        ______________________________________                                        Cell-free broth                                                                              -20° C.                                                                              >6     Months                                    Cell-free broth                                                                              22° C. 1      Day                                       Cell-free broth                                                                               4° C. 23     Days                                      Cell-free broth, Argon                                                                       22° C. 12     Days                                      Cell-free broth, Argon                                                                        4° C. 38     Days                                      Cell-free broth                                                                              22° C. 10     Days                                      50% Glycerol                                                                  Cell-free broth                                                                               4° C. 60     Days                                      50% Glycerol                                                                  Cell-free broth                                                                              -20° C.                                                                              >1     Year                                      50% Glycerol                                                                  Cell-free broth                                                                              22° C. 20     Days                                      50% Glycerol, Argon                                                           Cell-free broth                                                                               4° C. >1     Year                                      50% Glycerol, Argon                                                           Cell-free broth                                                                              -20° C.                                                                              >1     Year                                      50% Glycerol, Argon                                                           Lyophilized    -20° C.                                                                              >1     Year                                      Lyophilized    22° C. >1     Year                                      Lyophilized    37° C. >1     Year                                      Lyophilized    45° C. >1     Year                                      ______________________________________                                    

PPP Production

The present invention, after achieving a large-scale tyrosinaseproduction, is then directed to a PPP production process without the useof fermentation. To produce PPP the tyrosinase is placed in a bufferedaqueous solution and then substrates are added. The PPP production isobserved by measuring the optical density (OD) of the reaction mixtureat 400 nm (OD₄₀₀) at various intervals of time. The OD₄₀₀ is directlyproportional to the concentration of PPP and is monitored to determinethe end of the enzymatic reaction so that the PPP and their derivativescan be harvested when the OD has leveled off (see FIG. 3). To achievemaximum PPP production, a number of parameters have been explored andtaken into consideration. These include: (1) a buffer system in whichthe tyrosinase exhibits the highest activity; (2) the temperature atwhich optimum synthesis occurs; (3) the tyrosinase concentration inrelation to the amount and type of substrates; (4) the optimum pH forenzymatic reaction; (5) the adequate reaction time; (6) the size ofreaction vessel in relation to oxygen exposure; and (7) the amount ofdissolved O₂ in reaction mixture. The most preferred combination ofthese parameters, for the substrate L-tyrosine, which gives rise to theproduction of high levels of PPP has been determined by the applicantsand is described in Example 2(a).

The PPP may also be produced using an in vitro enzymatic method in avariety of organic solvents (see Examples 4(a), 4(b) and 4(c)). Thetyrosinase is stable and found to be reactive to the added substrates invarious organic solvents. The preferred organic solvent system isselected from, but not limited to, the group consisting of methanol,propanol, DMSO, and ethanol.

As described previously, the chemical and physical properties of the PPPproduced vary with the composition of substrates and the reactionconditions. Traditionally, the substrates used for PPP production aretyrosine, dopa or their derivatives. In the present invention PPP isproduced using tyrosinase mediated polymerization of tyrosine, and/ortyrosine derivatives, and/or tyrosine-containing oligopeptides,including tyrosine dipeptides and/or hydrolyzed proteins. When anoligopeptide is used in combination with l-tyrosine as the substrate,the color of the resulting PPP can be further modified by varying theratio of oligopeptides to tyrosine in relation to tyrosinaseconcentrations. The physical characteristics such as UV absorptionspectra, molecular weight, and solubility are also varied with thereaction and with the oligopeptide:tyrosine combination as well as theratio of such a combination (see Figures (see Examples 3a and 3b) forthe variation of UV absorption due to the changes of the combination andthe ratio of such combination). Consequently, as illustrated in thefollowing examples the desired physical and/or chemical characteristicsof PPP including but not limited to color can be obtained bymanipulating: (1) the oligopeptide:tyrosine combination; (2) the ratioof such a combination; (3) the tyrosinase concentration corresponding toeach combination; (4) the tyrosinase concentration corresponding to eachratio of one particular combination; (5) reaction conditions; and (6)the solvent system used.

A preferred oligopeptide:tyrosine combination used in the presentinvention comprises a combination of dipeptide:tyrosine wherein thedipeptide can be a naturally occurring or a chemically modifieddipeptide. Through the use of these substrates and l-tyrosinecombinations, it is possible to alter the chemical properties of the PPPas well as the physical properties including the colors of the PPP whichinclude, but are not limited to, red, blue, green, black, brown, orange,violet and yellow.

Chemical Modification Using Oxidants

Chemically changed PPPs can also be obtained by oxidation of the PPPswith an oxidant such as hydrogen peroxide. For example, l-tyrosinederived PPP (which is black in color) can be modified to a lighter colorby hydrogen peroxide (see Example 2b). The final color of PPP is afunction of the concentration of hydrogen peroxide used, the length oftime the PPP is oxidized, the pH and temperature. Changes in the UV andvisible spectra can be used to follow the rate of PPP oxidation andhydrogen peroxide can then be removed by ultrafiltration or passage ofthe reaction mixture through a catalyst such as platinum.

PPP Analyses

PPPs made by the methods described in the following examples weresubjected to some or all of the analytical procedures listed:

(1) UV and visible spectrophotometric scan.

(2) Molecular size determinations via membrane filtration or denaturingagarose electrophoresis.

(3) Color determination via spectrophotometric analysis to obtain L*a*b*values.

EXAMPLE 2(a) In Vitro Synthesis Production of PPP in Aqueous Solution

L-tyrosine melanin was synthesized in vitro with the Streptomycestyrosinase. A 3.0 L reaction containing 30 g L-tyrosine, 2610 ml 0.05MNa₂ HPO₄, 260 ml 0.05M NaH₂ PO₄, pH 8.3, 90 ml ultra-filtered,Streptomyces tyrosinase (40,500 dopachrome units), was performed in a6.0 L, Wheaton Proteus integral fermenter. The melanin synthesis wascarried out at 30° C., 400 RPM, and 1 liters per minute air flowsupplemented with 600 ml oxygen per minute. The RPM was increased to500, at 1 hour. Additional diafiltered tyrosinase was added at 2 hours,(20,250 units), 3.25 hours, (5,400 units), and at 3.5 hours, (20,250units). A final of 2,880 units tyrosinase per g L-tyrosine was achievedby this enzyme addition scheme. The rate of melanin synthesis wasfollowed by monitoring the increase in absorbance at 400 nm, (see FIG.3). An average rate of melanin synthesis of 1.76 g melanin/hour wasobtained in this reaction (1.0 g melanin is equivalent to 15-20 OD400units). Melanin synthesis was determined complete at 6 hours when therewas no significant increase in the OD400. Melanin was isolated after invitro synthesis by reducing the reaction mixture to a pH below 4.0 withHCl and recovering the precipitated melanin by centrifugation.Alternatively, the synthesized melanin can be chemically modifieddirectly in the reaction mixture.

EXAMPLE 2(b) Chemical Modification of In Vitro Synthesized PPP UsingHydrogen Peroxide

PPP produced as in example 2(a) was chemically modified by oxidationwith hydrogen peroxide. To 3 L of in vitro synthesized L-tyrosine PPP(in a 6 L Wheaton Proteus integral fermenter), 30% hydrogen peroxide wasadded until a final concentration of 3.3% hydrogen peroxide wasobtained. The PPP was oxidized at 30° C. and 350 RPM. The chemicallyoxidized PPP was recovered at various time points by acid precipitation,centrifugation and drying. The pH of the oxidized PPP solution wasreduced to below pH 2.5 by the addition of concentrated HCl. Theacidified PPP was placed at 4° C. for 17 hours to facilitateprecipitation. The precipitated PPP was then recovered bycentrifugation, 7,000 XG for 10 minutes. Residual amounts of hydrogenperoxide were removed by drying the melanin at 65° C. PPP was removedfrom the reaction mixture at various times and was analyzedspectrophotometrically. L*a*b* values were determined for each timepoint (see Table 2).

In the above example, hydrogen peroxide may be removed from the reactionmixture by ultra-filtration through spiral membranes. By choosing thespiral membrane's molecular weight cut-off, not only can the oxidizedmelanin be concentrated and the hydrogen peroxide removed, but specificsize fractions of melanin can be isolated.

                  TABLE 2                                                         ______________________________________                                        Oxidized PPP L*a*b* values                                                    Time (hours)                                                                              L*           a*     b*                                            ______________________________________                                        0           84.085       1.991   5.892                                        0.25        86.312       1.882   7.032                                        0.5         87.969       2.222  11.986                                        1.0         88.919       2.141  12.773                                        4.25        90.178       1.790  14.898                                        ______________________________________                                    

EXAMPLE 2(c) Methods of Removal of Hydrogen Peroxide and Recovery of PPPand PPP

Hydrogen peroxide was removed from the PPP and the PPP recovered byusing several methods: (1) PPP was acid precipitated by reducing the pHof the reaction mix to 1.5 by the addition of HCl. PPP was thenrecovered by centrifugation and the remaining hydrogen peroxide wasremoved by drying the PPP at 70° C.; or (2) hydrogen peroxide wasremoved from the reaction mixture by ultrafiltration using a 10,000 mwcutoff membrane. The PPP was then recovered by acid precipitation andcentrifugation as described in (1); or (3) hydrogen peroxide was removedfrom the reaction mixture by passage through a platinum coated material.Platinum causes hydrogen peroxide to degrade into water and oxygen. ThePPP was then recovered by acid precipitation and centrifugation asdescribed in step (1).

EXAMPLE 2(d) Chemical Modification of In Vivo Polyphenolic Polymers withH₂ O₂

Approximately 300 mg of L-tyrosine polyphenolic polymer produced in vivowas suspended in 60 ml of 0.2N NH₄ OH containing 1% H₂ O₂. The solutionwas incubated, with stirring, at room temperature for one hour. At thispoint 100 mg of platinum black was added and incubated at roomtemperature for an additional one hour.

The solution was filtered through a Whatman #1 filter and the pHadjusted to 3.0 with Concentrated HCl. The solution was Centrifuged andthe pellet was resuspended in either phosphate buffer at pH 7.0 or in0.01N NaOH.

EXAMPLE 2(e) Larger Scale Chemical Modification of In Vivo PPP with H₂O₂

One gram of dried polyphenolic polymer produced in vivo was stirred in40 ml of 0.1N NaOH for approximately one hour at room temperature.Hydrogen peroxide stock was prepared in 0.2N NaOH for a finalconcentration of 2%. To the suspended material, 40 ml of 2% H₂ O₂ /0.2NNaOH was added and stirred for 20 minutes at room temperature. Slowly, 7mg of platinum black was added to the mixture. The solution was thencentrifuged at 10,000 rpm for 10 minutes to remove the catalyst.Concentrated HCl was added to the supernatant to adjust the pH to 3.0.The solution was allowed to precipitate for approximately 2 hours at 4°C. After precipitation, the solution was centrifuged at 10,000 rpm for30 minutes. The precipitated sample was washed three times with 30 mlvolumes of acidified distilled water at pH 3.0. Finally, the sample waswashed once with 80 ml of 100% acetone and dried.

EXAMPLE 2(f)

Chemical Modification of In Vivo PPP: Removal of H₂ O₂ viaUltra-filtration

Ten grams of dry L-tyrosine polyphenolic polymer, produced in vivo, wassuspended in 400 ml of 0.2N NH₄ OH. To this mixture was added 400 ml of2% H₂ O₂ in 0.2N NH₄ OH and the solution was stirred for 20 minutes. ThepH was adjusted to 5.0 with 1N HCl and centrifuged at 10,000 rpm for 10minutes. The precipitated impurities were discarded and the supernatantcontaining the modified PPP was purified by ultra-filtration using a10,000 molecular weight cut-off membrane. Ultra-filtration continueduntil no residual hydrogen peroxide was detected. The peroxide freesolution was acidified to pH 3.0 with 1N HCl and the PPP was allowed toprecipitate for 2 hours at 4° C. and recovered by centrifugation at10,000 rpm for 20 minutes. The precipitate was washed three times with800 ml volumes of 0.001 n HCl followed by a wash with 800 ml of 100%acetone. The PPP was then dried in a vacuum oven at 37° C. forapproximately 16 hours.

EXAMPLE 3 (a)

In Vitro Production of PPP Using Dipeptides With or Without L-Tyrosine

The purpose of these experiments was to determine the feasibility ofproducing PPP using dipeptides in combination with l-tyrosine.

Procedurally, the detailed experimental steps are described as follows:500 ml flasks containing 50 ml of 50 mM sodium phosphate buffer, pH 8.0were prepared. The desired combinations of dipeptide is added totyrosine, followed by the addition of 250-500 units of Streptomycestyrosinase enzyme. For example, different PPPs are produced by thepresent invention wherein the ratios of the substrate/l-tyrosinecombination can be described by the following formula: "X/tyr (A/B),wherein (A/B) is the weight ratio between X (the substrate other thanl-tyr) and tyr", and said weight ratio is not required to be kept as aconstant for each and every substrate/l-tyrosine combination provided bythe present invention. Instead, a preferred embodiment of this inventionis to provide a variety of weight ratios for each substrate/l-tyrosinecombination. The substrate/l-tyrosine weight ratio combination isselected from, but is not limited to, a group consisting of tyr-ala/tyr(0.1/0.25), tyr-ala/tyr (0.2/0.25), tyr-ala/tyr (0.5/0.25), tyr-ala/tyr(1.0/0.25), phe-tyr/tyr (0.5/0.1), phe-tyr/tyr (0.2/0.05), phe-tyr/tyr(0.3/0.1), phe-tyr/tyr (0.2/0.1), and phe-tyr/tyr (0.1/0.1). The flasksare then placed at 25°-30° C. shaking at 300 rpm overnight. Theovernight reaction is decanted into 50 ml centrifuge tubes and 0.5 ml ofconcentrated HCl was added to each tube and mixed. The reaction mixtureis allowed to stand at 4° C. for 1-2 hours to completely precipitate thePPP out of the solution. The precipitated material is spun in a tabletop centrifuge at 3000-4000 rpm for 15 minutes. The supernatant wasdecanted and the PPP pellet is placed at 70° C. until completely dry, atwhich point the dry-weight of each sample was determined. Samples werethen subjected to further analysis as described previously. The resultsare shown in Table 3 and FIG. 4. The results shown in Table 3 and FIG. 4indicate a difference in the quantitative physical characteristic ofPPPs made from the same substrate/tyrosine combination but withdifferent ratios. For example, the molecular weights for the tyr-ala/tyrcombination were significantly changed when the ratio of the combinationchanged from 0.1-0.2/0.25 to 0.5-1.0/0.25. The UV spectrum and theL*a*b* values were also altered when the ratio of the combination wasvaried. Consequently, different colored PPPs were produced by varyingthe ratio of the substrate/l-tyrosine combination.

Likewise, as an example, different PPPs are produced from phe-tyr/tyrwherein this combination has different ratios and reacts with differenttyrosinase concentrations described by the following formula: "A/B ratioat Y units of tyrosinase". For example, different PPPs are formed byusing 0.3/0.1 at 500 units, 0.3/0.1 at 250 units, and 0.2/0.5 at 500units (FIG. 5 and Table 4).

                                      TABLE 3                                     __________________________________________________________________________            AMT (gms)/                                                                           MOL. WEIGHT                                                    SUBSTRATE                                                                             50 ml  (DALTONS)                                                                              L*  a*   b*                                           __________________________________________________________________________    Tyrosine                                                                              0.25   Insoluble                                                                              90.903                                                                            1.119                                                                              7.557                                        Tyr--Ala/Tyr                                                                          0.1/0.25                                                                             30K-200K 89.625                                                                            2.094                                                                              15.835                                       Tyr--Ala/Tyr                                                                          0.2/0.25                                                                             14K-200K 91.841                                                                            1.568                                                                              14.576                                       Tyr--Ala/Tyr                                                                          0.5/0.25                                                                             >3K-30   94.104                                                                            1.022                                                                              12.629                                       Tyr--Ala/Tyr                                                                          1.0/0.25                                                                             >3K-14   95.458                                                                            0.457                                                                              11.251                                       Phe--Tyr/Tyr                                                                          0.5/0.1                                                                              >3K-14   98.739                                                                            -0.076                                                                             3.391                                        Phe--Tyr/Tyr                                                                          0.2/0.05                                                                             N.T.     95.359                                                                            0.387                                                                              8.415                                        Phe--Tyr/Tyr                                                                          0.3/0.1                                                                              N.T.     97.293                                                                            0.015                                                                              6.593                                        Phe--Tyr/Tyr                                                                          0.2/0.1                                                                              N.T.     96.821                                                                            0.139                                                                              6.845                                        Phe--Tyr/Tyr                                                                          0.1/0.1                                                                              N.T.     93.269                                                                            0.902                                                                              10.438                                       Val--Tyr/Tyr                                                                          0.5/0.125                                                                            >3K-22K   96.35                                                                            1.353                                                                              7.382                                        __________________________________________________________________________

                  TABLE 4                                                         ______________________________________                                                   Amount               REACTION                                      SUBSTRATE  g/50 ml   ENZYME [ ] COLOR                                         ______________________________________                                        PHE--TYR/TYR                                                                             .3/.1     500 Units  Dark Green                                    PHE--TYR/TYR                                                                             .3/.1     250 Units  Dark Green                                    PHE--TYR/TYR                                                                             .2/.05    500 Units  D-Brown/Black                                 PHE--TYR/TYR                                                                             .2/.05    250 Units  D-Green/Brown                                 ______________________________________                                    

EXAMPLE 3(b) In Vitro Production of PPP via the Addition of Dipeptideswith Naturally Occurring and/or Biochemically/Chemically Modified AminoAcids

The results shown in Table 5 indicate that different dipeptides wereuseful substrates for the production of PPP with distinct physicalproperties, for example, reproducible colors. This is true whether thedipeptides consisted of naturally occurring amino acids, chemicallymodified amino acids, or a combination. The constituents of PPPdetermined the molecular weight, solubility, yield, UV spectrum, cost ofproduction and the ultimate colors of the PPPs. It is noted that blackPPPs were produced when a variety of hydrolyzed proteins, instead ofoligopeptides, were used as the substrates.

                                      TABLE 5                                     __________________________________________________________________________    SUBSTRATE     GRAMS/50 ml                                                                           REACTION COLOR                                                                           MOL WEIGHT                                   __________________________________________________________________________    Tyrosine      .25     Black                                                   Tyr--Ala      .05     Brown                                                   Tyr--Ala      1       Brown      >3000-14000                                  Tyr--Ala/Tyr  .05/.25 Dark-Brown                                              Tyr--Ala/Tyr   .1/.25 Dark-Brown   30000->200000                              Tyr--Ala/Tyr   .2/.25 Dark-Brown  14000-200000                                Tyr--Ala/Tyr   .5/.25 Dark-Brown >3000-30000                                  Tyr--Ala/Tyr    1/.25 Dark-Brown >3000-14000                                  Val--Tyr/Tyr   .5/.125                                                                              Red Brown  >3000-22000                                  N-Acetyl-L-Cysteine/Tyr                                                                     .5/.1   Yellow                                                  N-Acetyl-L-Tyrosine/Tyr                                                                     .5/.1   Red Brown  >3000-30000                                  Glycyl-L-Tyrosine/Tyr                                                                       .5/.1   Red Brown    14000->200000                              Phe--Tyr/Tyr  .5/.1   Green      >3000-14000                                  Ala--Tyr/Tyr  .5/.1   Brown                                                   L-Tyrosine Methyl                                                                           .5/.1   Yellow                                                  Ester/Tyr                                                                     L-Tyrosine Ethyl                                                                            .5/.1   Yellow                                                  Ester/Tyr                                                                     5-Hydroxydopamine/Tyr                                                                       .5/.2   Dark-Brown                                              L-3-Methoxy Tyrosine/Tyr                                                                    .5/.2   Black        22000->200000                              N-Acetylglycine/Tyr                                                                         .5/.2   Black                                                   L-Tyrosine Allyl                                                                            .5/.2   Yellow                                                  Ester/Tyr                                                                     3-Amino-L-Tyrosine/Tyr                                                                      .5/.2   Yellow                                                  Catachol/Tyr  .5/.2   Grey Black                                              3-Iodo-L-Tyrosine/Tyr                                                                       .5/.2   Black                                                   Tyr--Tyr/Tyr  .5/.2   Dark-Brown   30000->200000                              6-Hydroxydopamine/Tyr                                                                       .5/.2   Black                                                   L-Tyrosine Hydrazide/Tyr                                                                    .5/.2   Dark-Brown                                              DL-M-Tyrosine/Tyr                                                                           .5/.2   Black                                                   N-Chloroacetyl-L-                                                                           .5/.2   Red Brown   >3000-<14000                                Tyrosine/Tyr                                                                  L-α-Methyl Dopa/Tyr                                                                   .5/.2   Black                                                   Chlorogenic Acid/Tyr                                                                        .4/.2   Black                                                   N-Acetyl-L-   .2/.1   Black        30000->200000                              Tyrosinamide/Tyr                                                              Poly-L-Tyrosine/Tyr                                                                         .2/.1   Black                                                   Trp--Tyr/Tyr  .13/.05 Black                                                   L-β-3,4-Dihydroxyphe.                                                                  .12/05  Yellow-Brown                                            Ala.Met.Ester/Tyr                                                             Tyr--Gly/Tyr  .07/.05 Black        22000->200000                              Silk Protein/Tyr                                                                            .5/.5   Black       10000-100000                                Silk Protein/Tyr                                                                             1/.5   Black       10000-50000                                 Silk Protein/Tyr                                                                            1.5/.5  Black      <10000-46000                                 Silk Protein/Tyr                                                                             2/.5   Black      <10000-40000                                 Silk Protein/Tyr                                                                            2.5/.5  Black      <10000-30000                                 Silk Protein/Tyr                                                                             3/.5   Black      <10000-30000                                 Wheat Protein/Tyr                                                                            1/.5   Black        30000->200000                              Soy Protein/Tyr                                                                              1/.5   Black       <3,000-200000                               __________________________________________________________________________

EXAMPLE 4(a) Production of PPP in Organic Solvent

The in vitro synthesis of PPP via Streptomyces tyrosinase in organicsolvent systems was empirically studied. A selection of solvents andtheir concentrations in aqueous buffer was established for reactivitywith tyrosinase.

One ml reactions were conducted with varying concentrations of solvents,to which tyrosinase enzyme and substrate (l-dopa or l-tyrosine) wereadded to determine reactivity. As shown in the following Table 6,tyrosinase was found to be reactive with l-dopa and l-tyrosine in avariety of organic solvents such as methanol, ethanol, DMSO andpropanol. The maximum enzyme reactivity was observed for solventconcentrations between 50-70%.

                  TABLE 6                                                         ______________________________________                                                               C         D                                                 A         B       L-DOPA    L-TYROSINASE                                  1   SOLVENT   % [ ]   REACTIVITY                                                                              REACTIVITY                                   ______________________________________                                         2   Methanol  50      ++++      +++                                           3             70      +++       ++                                            4             90      +         --                                            5             100     --        --                                            6   Propanol  50      ++++      +++                                           7             70      ++++      +++                                           8             90      +++       +                                             9             100     --        --                                           10   DMSO      50      +++       +                                            11             70      --        --                                           12   Ethanol   50      ++++      +++                                          13             70      ++++      ++                                           14             90      ++        --                                           15             100     --        --                                           ______________________________________                                         In Table 6 a "dash" (--) symbol indicates no apparent reactivity. The         "plus " (+) symbols indicate visible reactivity between substrate and         tyrosinase. Thus going from lightly tinted (+) to black PPP (++++).      

EXAMPLE 4(b)

In Vitro Production of Polyphenolic Polymers in Organic Solvents

The following example describes steps followed to enzymatically produceL-tyrosine polyphenolic polymers in ethanol.

A two liter erlenmeyer flask containing 1.5 liters of 25 mM NaPO₄buffer, pH 7.0/40% ethanol was prepared. An amount of 7.5 grams ofL-tyrosine was added to the flask for a final concentration of 0.5%. Theenzyme tyrosinase (38,000 units) was added and the whole mixture wasplaced in an incubator at 25°-30° C. and shaken at 300 rpm forapproximately 48 hours.

Upon completion of incubation unincorporated L-tyrosine was removed bypassing the solution first through Whatman #1 filter and then through a0.8/μm filter. Concentrated hydrochloric acid was added to the filtrateto adjust the pH to 1.0. The solution was placed at 4° C. overnight andthen was centfifuged at 7,000 rpm for approximately 15 minutes. At thispoint two species of polyphenolic polymers were isolated: an acidprecipitable and an acid soluble fraction. The acid soluble fraction(supernatant) was decanted into a 4 liter beaker with a large bottomsurface area for efficient drying. Both the acid precipitable andsoluble fractions were then placed at 70° C. and dried.

Further sample preparation of the dried, acid soluble fraction wasperformed to obtain two additional and distinct species of polyphenolicpolymers. The dried acid soluble fraction was resuspended inapproximately 50 ml of 50 mM NaPO₄ buffer at pH 8.0. The pH of thesolution was then adjusted with NaOH to pH 7.0. At pH 7.0, a brown/tanprecipitate was formed. The mixture was centrifuged at 4,000 rpm for 15minutes and separated into two fractions, a precipitate and a solublefraction.

EXAMPLE 4(c) In Vitro Production and Oxidation of PPP in OrganicSolvents

An in vitro enzymatic PPP synthesis was conducted as follows: One literflasks were prepared which contained either 500 ml of 50% ethanol in 25mM sodium phosphate buffer at pH 8.0 or 500 ml of 25 mM sodium phosphatebuffer at pH 8.0. To each flask was added 1% L-tyrosine and 10,000 unitsof tyrosinase. The flasks were then placed on a shaker at 30° C. and 300rpm for 3 hours. The pH of each flask was adjusted to 9.0 by theaddition of NaOH. Hydrogen peroxide was added to the reaction mixturesso that its final concentration was 2%. The flasks were stirred at roomtemperature for 3.5 hours. Aliquots of 25 ml were removed at varioustime points for analysis. The pH of the reaction mixtures were adjustedto 2.0 with HCl which precipitated the modified PPPs. After storage at4° C. overnight, the mixtures were centrifuged and the resulting PPPswere dried and characterized as shown below in Table 7.

                  TABLE 7                                                         ______________________________________                                                  50% ETOH       CONTROL                                              ______________________________________                                        Molecular Size:                                                               (Reaction Time)                                                               t= 0        insoluble        insoluble                                        t = 15 min  50K-<10K         >200K->10K                                       t = 3.5 hours                                                                             50K-<10K (much lighter)                                                                        100K-<10K                                        Dry Yield:                                                                    (per 500 mls)                                                                 t= 0        0.4 gms          1.4 gms                                          t = 15 min  0.2 gms          n.d.                                             t = 3.5 hours                                                                             0.1 gms          0.7 gms                                          ______________________________________                                    

Streptomyces antibioticus incorporating the plasmid pBS1082S shown inFIG. 1 of this application deposited at the American Type CultureCollection (ATCC), Rockville, Md., USA, on Nov. 25, 1992 under the termsof the Budapest Treaty on the International Recognition of the Depositof Microorganisms for the Purposes of Patent Procedure and Regulationsthereunder (Budapest Treaty) and is thus maintained and made availableaccording to the terms of the Budapest Treaty. Availability of thisculture is not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

The deposited culture has been assigned ATCC deposit number 69132.

While the invention has been disclosed by reference to the details ofpreferred embodiments, the disclosure is intended to be illustrativerather than limiting, as it is contemplated that modifications willreadily occur to those skilled in the art, within the spirit of theinvention and the scope of the appended claims.

We claim:
 1. An in vitro method for producing polyphenolic polymers("PPP") comprising the steps of:producing extracellular recombinanttyrosinase in a microorganism selected from the group consisting ofStreptomyces, Escherichia, Bacillus, Streptococcus, Salmonella,Staphylococcus, and Vibrio, wherein said microorganism contains anexpression vector with polynucleotides encoding tyrosinase, andmicroorganism is grown in the presence of 20-90% dissolved oxygen in alow casein containing medium as nitrogen source, wherein the casein isselected from the group consisting of casein, casein hydrolysate orcasein peptone and mixtures thereof, and said tyrosinase is harvestedwhen the extracellular tyrosinase activity, in units/ml, stopsincreasing; reacting tyrosinase substrates with said tyrosinase to formPPP; and recovering the PPP so produced.
 2. The method of claim 1wherein the tyrosinase recovered when the extracellular tyrosinaseactivity reaches up to about 60 units/ml or more.
 3. The method of claim1 wherein said tyrosinase and said tyrosinase substrate are reacted inan organic solvent system.
 4. The method of claim 3 wherein said organicsolvent is selected from the group consisting essentially of methanol,propanol, ethanol, and DMSO.
 5. The method of claim 1 wherein the colorof said PPP is controlled by the amount of tyrosinase present relativeto the amount of tyrosinase substrate present.
 6. The method of claim 1wherein the dissolved-oxygen concentration is controlled during theproduction of ppp.
 7. The method of claim 1 wherein the color of the PPPis controllably altered by steps comprising:combining the PPP with astrong oxidizing agent; allowing the PPP to react with the strongoxidizing agent until the PPP attains the desired color; and removingthe oxidizing agent from the PPP.
 8. The method of claim 7 wherein saidoxidizing agent is selected from the group consisting essentially ofhydrogen peroxide, potassium permanganate, peracetic acid, andperchloric acid.
 9. The method of claim 7 wherein said oxidizing agentis removed from said PPP by ultrafiltration.
 10. The method of claim 7further comprising fractionating said oxidized PPP using selectedmolecular weight cut-off ultrafiltration.
 11. The method of claim 1wherein said expression vector is pBS1082S.
 12. The method of claim 1wherein said PPP is produced at about Ph 8.3.
 13. A method for producingpolyphenolic polymers ("PPP") comprising the steps of:producingextracellular recombinant tyrosinase in a microorganism selected fromthe group consisting of Streptomyces, Escherichia, Bacillus,Streptococcus, Salmonella, Staphylococcus, and Vibrio, wherein saidmicroorganism contains an expression vector with polynucleotidesencoding tyrosinase, and microorganism is grown in the presence of20-90% dissolved oxygen in a low casein containing medium as nitrogensource, wherein the casein is selected from the group consisting ofcasein, casein hydrolysate or casein peptone and mixtures thereof, andsaid tyrosinase is harvested when the extracellular tyrosinase activity,in units/ml, stops increasing; reacting tyrosinase substrates with saidtyrosinase to form PPP; reacting said tyrosinase with said tyrosinasesubstrate in an organic solvent system selected from at least one of thegroup consisting of: methanol, propanol, ethanol, and DMSO to form PPP;controlling the dissolved oxygen in said solvent during PPP production;and recovering the PPP so produced.
 14. The method of claim 13 whereinthe color of said PPP is controlled by the amount of tyrosinase presentrelative to the amount of tyrosinase substrate present.